FOB 8 DNA replication

• central dogma of dna

  DNA gives rise to new DNA via replication, it is copied to RNA via transcription and RNA can be copied to DNA via reverse transcription. RNA is translated into protein via translation.
• dna replication


  need when copying DNA to transfer to progeny (descendant) cells. we know more about prokaryotic system of E.coli than eukoryotes
• cell cycle


  G0, terminally differentiated cells withdraw from cell cycle indefinitely, and re-enter in G1. G1, RNA and protein synthesis, no DNA synthesis (6-12 hrs), contains a restriction point to commit to S phase. S, DNA synthesis doubles the amount of DNA in the cell, RNA and protein synthesized 6-8hrs. G2, no DNA synthesis, RNA and protein synthesis continues 3-4 hrs. M phase, mitosis (nuclear division) and cytokinesis (cell division) yield two daughter cells 1hr. can to G0 or G1 again.
• summary of replication


  begins at the origin of replication, occurs in replication forks, old DNA strand acts as a template for the synthesis of a new strand, semi-conservative process, mediated by proteins (enzymes and DNA binding proteins). strands split apart and old strands act as templates to create new strands. at origin of replication DNA is replicated towards 5 prime direction, and strands in reverse are made in fragments
• replication (intense)


  DnaA binds OriC twisting AT sections opens duplex. DnaC loads Helicase DNaB into fork separates DNA(replication bubble). DNA gyrase binds preventing supercoiling. single-strand binding proteins SSB bind to ssDNA preventing digestion, premature annealing and 2* structures. primase inserts RNA primer, polymerase binds. DNA polymerase III synthesises complimentary strand 5-3, from template. leading continuously, lagging discontinuously okazaki fragment. RNase H remove primers, replacement DNA by DNA polymerase I. DNA ligase join okazaki. Finishes at termination sequence Ter
• models of dna replication


  conservative, semiconservative and dispersive. describe what is done with the original and new strand of DNA. conservative, original strands are copied and two old strands are put together in new cell. semi conservative is two old strands are split, copied and each set has one old and one new strand. dispersive is bit of old and new chunks, disproven quickly. e.coli is semi-conserved
• DnaA

  binds to origin of replication (oriC) initiates replication in e.coli
• DnaB

  helicase. unwinds DNA helical structure
• DnaC

  required for DnaB binding at origin
• SSB

  single stranded binding proteins. bind to single stranded DNA preventign re-annealing, degradation and formation of secondary structures
• DnaG

  primase. synthesises short RNA primers to initiate replication. DNA polymerase can only add nucleotides to a pre-existing 3' OH group
• DNA gyrase

  topoisomerase II. reduces tosional strain (supercoiling) that occurs during DNA unwinding
• DNA polymerase III


  catalyses elongation of the growing (new) DNA strand through the addition of nucleotides (dNTP) to the free 3' OH end 5'->3'
• RNase H or equivalent


  RNA endonuclease that removes (cleaves RNA primers)
• DNA ligase


  catalyses the joining (ligation) of two DNA segments
• initiation factors


  HU, FIS, IHF stimulate initiation of replication
• replication forks are


  bidirectional. opened for as short as possible because it cannot be used when replicated. better than unidirectional.
• three phases of replication

  initiation (regulated beginning), elongation (synthesis of new strands) and termination (end)
• initiation


  oriC (e.coli 245 bp region) binding site for DnaA, causing totional strain at an area rich with A=T called DNA unwinding element (DUE), which opens the helix and separates DNA strands. Helicase DNaB is loaded onto the separated DNA strands with the aid of DnaC. DNaB unwinds the strands and produces 2 replication forks (bidirectional). DNA gyrase (topoisomerase II) relieves torsional strain (supercoiling) SSB bind to ssDNA stabilising them. initiation complete when DNA gyrase, helicase, SSB and DnaA have bound and opened DNA
• elongation


  primase creates small RNA primer to initiate replication, 10-12 nucleotides long. DNA polymerase binds the RNA primer and elongates a new reverse complementary DNA strand either in a continuous (leading) or non-continuous (lagging strand) manner in 5-3 direction. polymerase can only add to prexisitnf free 3' OH end.
• continous process


  leading strand DNA polymerase III binds RNA primer and elongates a new DNA strand continuously in a 5-3 direction as the DNA is unwound ahead. the one rna primer used is removed
• non-continuous elongation


  lagging strand, moving towards the replication fork, the new DNA strand would be synthesised in a 3-5 direction. DNA polymerase can only synthesise in 5-3 direction. therefore the new DNA strand is made in okazaki fragments. prokayrote (1,000-2,000 nucleotides long) eukaryotes (100-200 nctd long). RNase H or equivalent and DNA polymerase I removes and replace RNA primers with DNA. DNA ligase joins fragments together
• how does DNA polymerase elongate


  DNA pol extends new strand by adding nucleotides from deoxynucleotide triphosphates dNTPs to the growing strand: dATP, dCTP, dGTP and DTTP (building blocks and energy). using complementary base-pairing rules DNA polymerase catalyses the formation of a phosphodiester bond between the free 3' hydroxyl OH group on the growing DNA strand and the dNTP a-phosphate. DNA pol requires 2 Mg2+ ions as cofactors the first facilitated the 3' oH group nucleophilic attack on the a phosphate. the second helps stabilise and displace the pyrophosphate (di-phosphate)
• lagging + leading strand synthesis


  two polymerases joined together, the lagging strand is looped to solve the antiparallel problem. this allows both polymerases to synthesise in the same direction at the same time.
• DNA polymerase III complex


  2 poll III core subunits linked to B sliding clamps liked by clamp loader. enzymatic activity occurs in dual-core units. one core unit synthesised the leading strand and one the lagging strand. clamp loading component loads B clamp increase processing rate and prevent dissociation from strands. clamp loader loads when there is a primer and detaches at termination sequences (leading) or Okazaki fragment ends.
• synthesis


  dna polymerase 3 clamps onto strand and core subunits with beta clamp latch onto laggng and leading strands. continuous synthesis on leading strand and okazaki fragments on looped lagging strand. the strand loops around and when the start of a previous fragment is reached the B clamp is discarded and a new B clamp is loaded for the new fragment
• termination


  in e.coli this is due to circular DNA, two forks meet and termination occurs at Ters. termination occurs in terminus regions 20bp sequences of repeats (Ter) which is a binding site for Tus (terminus utilisation substance) proteins. A tus-Ter complex will trap one replication fork and the other fork will halt when it meets the first arrested fork. many different Ter proteins (A,B,C etc) that can stop clockwise or counterclockwise. this leads to 2 interlinked rings of DNA, topoisomerase IV separates the 2 DNAs by unwinding both, cutting one, pulls the other ring through and reseals
• ensuring fidelity


  the sequences must be correct, mistakes can affect or eliminate the function of genes. DNA polymerases copy nucleotide sequences with speed and fidelity (mistake once every 10^9 or 10 nucleotides). detection of mistakes relies on hydrogen bonding and geometry (only A=T C=-G have correct geometry). DNA polymerase has 3'->5' exonuclease activity (double checks and removes mismatch mutations), incorrect insertion of nucleotide inhibits further elongation, allowing 3'->5' exonuclease activity to take place
• eukaryotes DNA replication


  more DNA, in nucleoprotein complexes (chromosomes with histone complexes (nucleosomes)), greater complexity and highly regulated in cell cycle, essential features of replication are the same. multiple origins of replications and initiate at the origin recognition complex ORC. polymerases are named differently and termination involves telomeres